Marc Plante, Bruce Bailey, and Ian Acwortth
Thermo Scientific, Chelmsford, MA, USA
Ap plica t ion Note 71 75 9
Analysis of Lipids by RP-HPLC Using
the Dionex Corona ultra
Key Words
Dionex Corona, Halo HPLC Column, CAD, lipids,
reversed-phase HPLC
Abstract
With the increasing interest in lipidomics, analytical
methods are required to quantify samples with high
sensitivity and selectivity. We have developed a 72-minute,
reversed-phase high pressure liquid chromatography
(HPLC) method, using the Thermo Scientific™ Dionex™
Corona™ Charged Aerosol Detector (CAD) in combination with a fused-core (Halo) C8 150 × 4.6 mm (2.7 µm)
HPLC column. This method has broad selectivity that
can separate and quantify lipids, containing a wide range
of hydrophobicity. Free fatty acids (lauric to stearic acid),
fatty acid-esters and alcohols (tetradecanol to docosol),
phospholipids (LPC, DPPC, DPPE, PE, PS, PC,
sphingomyelin), acylated glycerols (mono-, di-, and
tri-acylglycerols and milk fats), and paraffins (octadecane
to octacosane) have been characterized using this single
method. Typical dynamic ranges cover approximately
three orders of magnitude contained within
(10–10,000 ng on column (o.c)) and limits of detection
(LOD) values are < 30 ng o.c.
Introduction
Lipids are a structurally diverse group of naturallyoccurring, water-insoluble compounds that can, for
convenience, be divided into the following eight
categories: fatty acyls (e.g., fatty acids), glycerolipids
(e.g., monoacylglycerides, diacylglycerides, triacylglycerides), glycerophospholipids (e.g., phosphatidyl choline,
phosphatidyl serine), sphingolipids, sterol lipids
(e.g., cholesterol, bile acids, vitamin D), prenol lipids
(e.g., vitamins E and K), saccharolipids and polyketides
(e.g., aflatoxin B1).1 Even within a particular category,
there can be great structural complexity. For example,
although triglycerides are composed of one glycerol
molecule and three fatty acid molecules, differences in
fatty acid chain length, degree of unsaturation, position of
unsaturation and position on the glycerol backbone, result
in numerous triglyceride variants.
Typically, much of the chromatography for lipids has been
performed using normal phase methods, where the solvent
is less polar than the stationary phase. Normal phase
HPLC separates largely on differences in polarity between
different analytes and their interactions with the stationary phase and solvents.2 The solubility of lipids in normal
phase solvents makes sample preparation simpler than
for reversed phase systems. However, certain solvent
combinations can allow for non-polar sample solvents to
be used in reversed phase chromatography, which offers
different selectivity than is achieved using normal phase.
In this presentation we show chromatography based on
reversed-phase HPLC combined with the Dionex Corona
CAD. CAD is a mass-dependent detector and responds
to all non-volatile analytes, independent of chemical
structure. Reversed-phase HPLC separates analytes based
on their different interactions with the solvents through
hydrogen bonding and dipole-dipole attractions. This
yields different selectivity than normal-phase HPLC.2
This method is capable of separating the relatively
hydrophilic steroids to the completely hydrophobic
paraffins using a single run even resolving analytes that
are closely related in structure. The Dionex Corona CAD,
with greater sensitivity over that of evaporative light
scattering detection, can also reveal analytes that may
not be detected by other means (e.g. mass spectrometry,
ultraviolet or flow injection).
Experimental
180
Retinyl Acetate
Vitamin K1
CoQ10
140
12
100
80
60
Corona ultra Parameters
Gas:
Lutein
160
Peak Area
In this presentation a number of examples are presented
including: analysis of free fatty acids, fatty alcohols, a
natural oil fingerprint, a tissue sample, as well as the
separation and quantitation of ten fat-soluble vitamins.
Acceptable calibration curves can be generated from data
obtained using this method. This allows for the quantitation of known components contained in complex
matrices. Sample preparation is simple, with dissolution in
methanol / chloroform, varying from a ratio of 1:1 to 1:3,
depending on the solubility of the sample matrix.
35 psi via nitrogen generator
40
Filter:None
Range:
500 pA
Nebulizer Heater:
30 °C
20
0
HPLC Parameters
Mobile Phase:
A) Methanol / water / acetic acid (750 : 250 : 4)
Mobile Phase:
B) Acetonitrile / methanol / tetrahydrofuran / acetic acid (500 : 375: 125 : 4)
General Lipids
Gradient:
Time
%A
0
50
100
150
200
Load (ng)
250
300
350
300
Trans-Retinol
Vitamin E-alpha
Vitamin E-delta
Lycopene
Vitamin E-gamma
Retinyl Palminate
250
Fat-soluble Vitamins
%B
Time
%A
%B
0.00
100.0
0.0
0.00
70.0
30.0
46.00
30.0
70.0
1.00
50.0
50.0
60.00
10.0
90.0
5.00
40.0
60.0
65.00
10.0
90.0
10.00
35.0
65.0
65.10
100.0
0.0
12.00
90.0
10.0
72.0
100.0
0.0
17.00
100.0
0.0
17.10
70.0
30.0
20.00
70.0
30.0
Flow Rate:
0.8 mL/min
1.5 mL/min
Run Time:
72 min
20 min
HPLC Column:
Halo C8, 150 × 4.6 mm, 2.7 µm
Column Temperature: 40 °C
45 °C
Sample Temperature: 0 °
10 °C
Injection Volume:
10 µL
10 µL
Sample Preparation
Samples were prepared by diluting 1 mg of analyte in 1
mL of methanol / chloroform (1:1 – 1:3). Extremely
hydrophobic samples were dissolved in 3 parts chloroform, with 1 part methanol added. Fat-soluble vitamins
were dissolved in ethanol/ butylated hydroxyanisole (10
mg/L) at stock concentrations of 100 or 1000 µg/mL.
Fat-Soluble Vitamins Calibration
Calibration plots of ten fat-soluble vitamins are shown in
Figure 1. Three vitamin supplements were extracted and
quantified, with the percent of label claim shown in Table 1.
200
Peak Area
2
150
100
50
0
0
50
100
150
200
Load (ng)
250
300
350
Figure 1. Calibration plots of ten fat-soluble vitamins. All relative standard deviations (RSDs) were < 5% for all amounts above 20 ng o.c. This indicates good
precision for the method results and LOD values were determined at less than
10 ng o.c.
All of the correlations, fit to second-order polynomials, resulted in coefficients
between 0.994–0.999.
Table 1. Vitamin supplements were extracted and quantified.
Vitamin
E-alpha
CoQ10
109.8%
N/A
72.7
Solgar VM-75®
97.2%
N/A
N/A
CVS® Vitamin E 400 IU
Capgels
N/A
97.6
N/A
Product
Whole Foods® CoQ10
200 mg
Vitamin E-alpha
succinate*
*Vitamin E-alpha succinate calculated based on Vitamin E-alpha correlation.
Examples
3
Tissue Lipids
Free Fatty Acids
Stearic Acid
Cholesterol
Palmitic Acid
Linoleic Acid
Oleic Acid
Lineonlic Acid
Phospholipids
Free Fatty Acids
Myristic Acid
Lauric Acid
Figure 2. Lauric to stearic acids, including unsaturated acids in methanol/chloroform (1:1),
100–11,000 ng on column. Greater response was found with larger-molecular weight
analytes, due to decreases in vapor pressure.
Figure 5. Extract from rat brain in methanol / chloroform (1:1), showing the expected components of fatty acids, phospholipids, and cholesterol. The phospholipid region is expanded in
the inset to show additional detail.
Fatty Alcohols
Fat-soluble Vitamins
Tetradecanol
Eicosanol
Docosanol
Octadecanol
Hexadecanol
Figure 3. Tetradecanol to docosanol in methanol/chloroform (1:1), 100–12,000 ng on
column. Greater response was found with larger-molecular weight analytes, due to decreases
in vapor pressure.
Stearic Acid
Peak #
CompoundPalmitic Acid LOQ (ng)
1
Trans-Retinol
4
Oleic
2 Linoleic
Retinyl
7 Acid
Acid Acetate
3
Lutein
10
4
Vitamin E- delta
5
Lineonlic
AcidVitamin E- gamma
5
6
6
Vitamin K1
15
Myristic Acid
7
Vitamin E- alpha
9
8
Lycopene
42
Lauric Acid
9
Retinyl Palmitate
15
10
CoQ10
7
1
9
5
4
2
Natural Oils
10
7
3
8
6
Free Fatty Acids
Steroids
Monoacylglycerides
Fatty Alcohols
Diacylglycerides
Triacylglycerides, Paraffins
Phospholipids
Sterols
Figure 4. Algal oil from hexane wash, dissolved in methanol/chloroform (1:1), 10 µg on
column. Lipid classifications were determined from other, independent standard analyses.
Figure 6. HPLC-CAD of ten fat-soluble vitamins, in ethanol/BHA (10 mg/L), each at 165 ng
on column. Lycopene (#8) exhibited fronting, which increased the limit of quantitation (LOQ).
Discussion and Conclusions
Vapor pressure can affect the sensitivity of some analytes,
as shown in the chromatograms for the free fatty acids
and fatty alcohols. As analyte vapor pressure increases,
fewer particles form which decreases the amount of
response. For the free fatty acids, the lowest molecular
weight compound that showed response is Lauric acid. To
improve response of acidic or basic semi-volatile analytes,
the addition of volatile buffer salts can transform
relatively volatile analytes into non-volatile salts, which
can then be determined with the Dionex Corona CAD.6
For a sample of unknown lipids, this method can be run
as an initial screen; gradient optimization can then be
used for analysis of a particular suite of lipids. The
method is also flexible: the gradient can be adjusted to
optimize for separation and run time, as was shown with
the vitamin sample analysis.
The method can be used for quantitative analysis.
Calibration curves were created for 10 fat-soluble
vitamins, each with a correlation coefficient > 0.994, fit to
second-order polynomials. For all analytes evaluated here,
the LOQ, based on S/N = 10, was < 10-45 ng on column,
which is lower than can be achieved with evaporative
light-scattering detectors (ELSD). Precision was acceptable, with RSDs < 7% across all amounts above 20 ng o.c.
and for all fat-soluble vitamins evaluated here. Percent
recovery values on vitamin products showed quantitative
results, with ~98% label claim for Vitamin Es and 74%
label claim for a CoQ10 sample was found, possibly
attributable to incomplete extraction of the latter from the
product matrix.
This method is routinely used to separate a wide variety
of lipids in many matrices, including milk, plant oils,
tissues, and vitamin supplements. Preliminary results
obtained through this initial screening process allow for
further chromatographic optimization to characterize the
specific lipids relevant to the sample.
References
1.Fahy, E., Subramaniam, S., Brown, A., Glass, C.,
Merrill, A., Murphy, R., Raetz, C., Russell, D., Seyama,
Y., Shaw, W., Shimizu, T., Spencer, F., van Meer, G.,
VanNieuwenhze, M., White, S., Witztum, J., and Dennis,
E. (2005). A comprehensive classification system for
lipids. J. Lipid Res., 46, 839–861. [Online] http://www.
ncbi.nlm.nih.gov/pubmed/15722563 (accessed Aug 14,
2015).
2.Venn, R.F., (2000). Principles and Practice of Bioanalysis. Taylor & Francis (United Kingdom), pages 79 and
87.
3.Lisa, M., Lynen, F., Holapek, M., Sandra, P. (2007).
Quantitation of triacylglycerides from plant oils using
charged aerosol detection with gradient compensation,
J. Chrom. A, 1176, 135–142. [Online] http://www.ncbi.
nlm.nih.gov/pubmed/18021788 (accessed Aug 14,
2015).
4.Yasuhiro, M., Eiko, M., Hiroshi, T., Masahide, Y.,
Toshihiro, O., Yoshikazu, S., Yuko, K., Nakao, I., and
Yoshihiko, I. (2002). Biological activity of 4-acetyltropolone, the minor component of Thujopsis dolabrata
SIEB. et ZUCC. hondai MA. Biol. Pharm. Bull., 25(8),
981–985. [Online] http://www.ncbi.nlm.nih.gov/
pubmed/12186430 (accessed Aug 14, 2015).
5.Katoh, T., Tanaka, R., Takeo, M., Nishide, K., and
Node, M. (2002). A new synthesis of a potent cancer
chemopreventive agent, 13-oxo-15,16-dinorlabda8(17),11E-dien-19-oic acid from trans-communic
Acid, Chem. Pharm. Bull., Vol. 50, 1625-1629. [Online]
http://www.ncbi.nlm.nih.gov/pubmed/12499606
(accessed Aug 14, 2015).
6.Ottosson, J. and Stefansson, M. (2006) “Detection of
non-volatile to volatile compounds by charged aerosol
detection (CAD)” Poster, Astra Zeneca (Sweden).
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Ap plica t ion Note 71 75 9
This method shows great selectivity for many different
lipid compounds, with sufficient dynamic range to
measure both major and minor constituents simultaneously (e.g. the natural oils chromatogram) and without
the need for additional standards when response factors
are shown to be consistent.3 The high resolution of
the chromatography enables the separation of many
compounds. The mobile phase is also compatible with
mass spectrometry, allowing for the direct identification
of these compounds by m/z ratios.3 This is especially
useful for drug discovery efforts, where minor components that are identified and isolated are often found to
possess biological activity,4 or may be very potent
pharmaceutical candidates.5